Method for Treatment of Constipation-Predominant Irritable Bowel Syndrome

ABSTRACT

The present invention provides methods for treating constipation-predominant irritable bowel syndrome comprising administering to a patient in need thereof, a polymeric proanthocyanidin composition from a  Croton  species or  Calophyllum  species in an amount sufficient to treat constipation-predominant irritable bowel syndrome (c-IBS). Treatment of c-IBS includes the treatment of the constipation component of c-IBS as well as the pain and abdominal discomfort associated with c-IBS. In one embodiment, the polymeric proanthocyanidin compound is crofelemer. The present invention in an alternative embodiment also provides methods for treating alternating constipation-predominant/diarrhea-predominant irritable bowel syndrome.

This application is a continuation under 35 U.S.C. § 120 of U.S. patentapplication Ser. No. 11/741,796, which claims benefit of priority toU.S. provisional patent application 60/797,076, filed May 1, 2006, thecontents of each of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Irritable bowel syndrome (IBS) is a common functional disorder of thebowel that has a pronounced effect on quality of life. A definingcharacteristic of IBS is abdominal discomfort or pain. The Rome IIDiagnostic Criteria (a system for diagnosing functional gastrointestinaldisorders based on symptoms) for IBS is as follows: at least 12 weeks ormore, which need not be consecutive, in the preceding 12 months ofabdominal discomfort or pain that is accompanied by at least two of thefollowing features: (1) it is relieved with defecation, and/or (2) onsetis associated with a change in frequency of stool, and/or (3) onset isassociated with a change in form (appearance) of stool.

Other symptoms that support the diagnosis of IBS include pain; abnormalstool passage (straining, urgency, or feeling of incomplete evacuation);passage of mucus; and bloating or feeling of abdominal distension.Patients can be sub-divided by their underlying bowel habits: (i)diarrhea-predominate IBS, (ii) constipation-predominate IBS, and (ii)constipation alternating with diarrhea (alternating IBS).

The pathophysiology of IBS is poorly understood despite the fact thatabout a quarter of the population in the UK may exhibit the symptoms,and approximately 15 percent of U.S. adults report symptoms that areconsistent with the diagnosis of IBS. It is estimated that only 25percent of persons with IBS seek medical care. In addition, patientsdiagnosed with IBS are at increased risk for other, non-gastrointestinalfunctional disorders such as fibromyalgia and interstitial cystitis.

IBS is the most common diagnosis made by gastroenterologists in theU.S., and accounts for 12 percent of visits to primary care providers.Approximately $8 billion in direct medical costs and $25 billion inindirect costs are spent annually in the U.S. for diagnosing andtreating IBS. Thus, IBS accounts for a large proportion of annualhealthcare costs in the U.S.

Primary treatment of IBS involves counseling and dietary modification.Drug therapy is considered to be beneficial if directed at individualsymptoms. For diarrhea predominant cases, antidiarrheal drugs such asloperamide can be used, which treat diarrhea, but not abdominal pain.Since abdominal pain is one of the defining characteristic of IBS,anti-diarrheal drugs do not adequately treat IBS (Jailwala et al., 2000,Ann Intern Med. 2000; 133:136-147; Cremonini et al., 2004, Minerva Med95:427-441). For constipation predominant cases, ispaghula is often usedto increase dietary fiber. Where patients have pain and distension aspredominant symptoms, anti-spasmolytics are commonly used. Mebeverineand peppermint oil are often used in such cases. Other agents that havebeen tried for treating IBS include beta-blockers, naloxone,ondansetron, calcium channel blockers, simethicone, leuprorelin,octreotide and cholecystokinin antagonists with variable results(Martindale, The Extra Pharmacopoeia, 31 st Edition (1996) p. 1197).

U.S. Pat. Nos. 5,211,944 and 5,494,661 to Tempesta disclose the use of aproanthocyanidin polymeric composition isolated from Croton spp. orCalophyllum spp. for the treatment of viral infections. Rozhon et al.,U.S. Patent Publication No. 2005/0019389, disclose the use of aproanthocyanidin polymeric composition isolated from Croton spp. orCalophyllum spp. for the treatment of secretory or traveler's diarrhea.Di Cesare et al., 2002, Am J Gastroenterol 10:2585-2588 disclose aclinical trial of crofelemer as a treatment for traveler's diarrheacompared to placebo. Dosages used in this study were 500 mg/day (125four times a day); 1000 mg/day (250 mg four times a day); and 2000mg/day (500 mg four times a day) for two days. The study showed that thecomposition was useful for the amelioration of stool frequency andgastrointestinal symptoms in patients with traveler's diarrhea.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF THE INVENTION

The present invention relates to methods for treating at least onesymptom of constipation-predominant irritable bowel syndrome (c-IBS) byadministering a composition or extract obtained from a Croton species orCalophyllum species. In one embodiment, the composition is a latex froma Croton species or Calophyllum species. In yet another embodiment, thecomposition is a polymeric proanthocyanidin composition from a Crotonspecies or Calophyllum species. Exemplary symptoms of c-IBS includepain; abdominal discomfort, such as abdominal fullness, bloating orswelling; abnormal stool frequency, i.e., fewer than three bowelmovements per week; hard or lumpy stools; straining during a bowelmovement; urgency and feeling of incomplete bowel movement. Thus, in oneembodiment, the invention provides a method for the treatment of one ormore symptoms of c-IBS comprising administering to a patient in need ofsuch treatment, an amount of a polymeric proanthocyanidin compositionfrom a Croton species or Calophyllum species effective to treat the oneor more symptoms of c-IBS. In preferred embodiments, the dosage of thecomposition is bioequivalent to orally administered enteric coatedcrofelemer at a dosage of about 250 mg per day to about 4000 mg per day.In one embodiment, bioequivalency is a sufficient dose of a compositionof the invention to produce the desired effects as seen with anothercomposition at a particular dosage, e.g., enteric coated crofelemer at adosage of about 250 mg per day to about 4000 mg per day. In anotherembodiment, bioequivalency is as defined by, or is as determined inaccordance with methods approved by, the U.S. Food and DrugAdministration. In a particular embodiment, the composition of theinvention is co-administered with an anti-inflammatory or analgesiccompound, which compound is preferably not systemically absorbed or ismodified such that the compound is not systemically absorbed. Suchcompounds include 5-ASA, sulfasalazine, mesalamine, APAZA, celecoxib androfecoxib.

In a particular embodiment, the present invention is directed to amethod of treating pain associated with c-IBS comprising administeringto a patient in need of such treatment, an amount of a polymericproanthocyanidin composition from a Croton species or Calophyllumspecies effective to treat pain associated with c-IBS. In anotherembodiment, the present invention is directed to a method of treatingabdominal discomfort associated with c-IBS comprising administering to apatient in need of such treatment, an amount of a polymericproanthocyanidin composition from a Croton species or Calophyllumspecies effective to treat abdominal discomfort associated with c-IBS.Optionally, analgesic or anti-inflammatory agents can be co-administeredwith the composition. In particular, the composition is formulated or ismodified such that it is not systemically absorbed, i.e., only 20%, 15%,10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less than 0.5% absorbed of the dosagegiven.

In yet another embodiment, the present invention is directed to a methodof treating abnormal stool frequency associated with c-IBS comprisingadministering to a patient in need of such treatment, an amount of apolymeric proanthocyanidin composition from a Croton species orCalophyllum species effective to treat the abnormal stool frequencyassociated with c-IBS, e.g., by increasing the number of stools perweek.

Exemplary polymeric proanthocyanidin compositions useful in the presentinvention are preferably isolated from a Croton species or Calophyllumspecies. For example, the composition can be a proanthocyanidin polymercomposition isolated from a Croton spp. or Calophyllum spp by any methodknown in the art. For example, the proanthocyanidin polymer compositionmay be isolated from a Croton spp. or Calophyllum spp. by the methoddisclosed in Example 2, infra, or disclosed in U.S. Pat. No. 5,211,944or in Ubillas et al., 1994, Phytomedicine 1: 77-106. PCT applicationPCT/US00/02687, published as WO 00/47062, discloses a method ofmanufacturing a proanthocyanidin polymeric composition isolated fromCroton spp. or Calophyllum spp. In preferred embodiment, the compositionis enteric protected, e.g., enteric coated. Thus, in a particularembodiment, the present invention is directed to a method of treatingabnormal stool frequency associated with c-IBS comprising administeringto a patient in need of such treatment, an amount of a polymericproanthocyanidin composition isolated from Croton spp. or Calophyllumspp. effective to treat the abnormal stool frequency associated withc-IBS, in which said amount is bioequivalent to an orally administereddose of about 250 mg per day to about 4000 mg per day of entericprotected crofelemer.

In one preferred embodiment, a proanthocyanidin polymer composition ofthe invention is crofelemer. Crofelemer (CAS 148465-45-6) is anoligomeric proanthocyanidin of varying chain lengths derived from theDragon's Blood of Croton lecheri of the family Euphorbiaceae. Crofelemerhas an average molecular weight of approximately 1900 daltons toapproximately 2700 daltons. The monomers comprising crofelemer comprisecatechin, epicatechin, gallocatechin, and epigallocatechin. The chainlength of crofelemer ranges from about 3 to about 30 units with anaverage chain length of about 8 units. Thus, in a preferred embodiment,the present invention is directed to a method of treating abnormal stoolfrequency associated with c-IBS comprising orally administering to apatient in need of such treatment, an amount of enterically-protectedcrofelemer effective to treat the abnormal stool frequency associatedwith c-IBS, in which said amount is between about 250 mg per day andabout 4000 mg per day. In certain embodiments where crofelemer isotherwise formulated (not enterically protected), the dosage ofcrofelemer administered is bioequivalent to a dosage of about 250 mg perday to about 4000 mg per day of orally administeredenterically-protected crofelemer. For example, such formulation is adelayed release formulation.

In an alternative embodiment, the present invention is directed to amethod for treating one or more symptoms of alternatingconstipation-predominant/diarrhea-predominant irritable bowel syndrome(a-IBS) comprising administering to a patient in need of such treatment,an amount of an extract or latex from a Croton species or Calophyllumspecies effective to treat the one or more symptoms of a-IBS. In oneembodiment, the composition is a polymeric proanthocyanidin compositionfrom a Croton species or Calophyllum species. In one aspect of thisembodiment, the present invention is directed to a method for treatingpain associated with alternatingconstipation-predominant/diarrhea-predominant irritable bowel syndrome(a-IBS) comprising administering to a patient in need of such treatment,an amount of a polymeric proanthocyanidin composition from a Crotonspecies or Calophyllum species effective to treat the pain associatedwith a-IBS. In preferred embodiments, the dosage of the composition isbioequivalent to orally administered enteric coated crofelemer at adosage of about 50 mg per day to about 4000 mg per day. In oneembodiment, bioequivalency is a sufficient dose of a composition of theinvention to produce a similar therapeutic effect as seen with anothercomposition at a particular dosage, e.g., enteric coated crofelemer at adosage of about 250 mg per day to about 4000 mg per day. In anotherembodiment, bioequivalency is as defined by, or is as determined inaccordance with methods approved by, the U.S. Food and DrugAdministration. Individuals suffering from a-IBS experience symptomsassociated with both c-IBS and d-IBS depending on the type of IBS thepatient is experiencing at a particular time. Thus, an a-IBS symptomthat can be treated by administering a composition of the inventionincludes, pain, abdominal discomfort, abnormal stool frequency, abnormalstool consistency, presence of urgency, diarrhea, and constipation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph illustrating the Effect of Crofelemer 125 mg bid onStool Frequency in Females.

FIG. 2 is a graph illustrating the Effect of Crofelemer 125 mg bid onUrgency in Females.

FIG. 3 is a graph illustrating the Effects of Crofelemer on AdequateRelief of IBS Symptoms in Females.

FIG. 4 is a graph illustrating the Effect of Crofelemer on Pain Score inFemales.

FIG. 5 is a graph illustrating the Effect of Crofelemer on Percent ofPain Free Days in Females.

FIG. 6 is a graph illustrating the Effect of Crofelemer on Pain Score inFemales.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for treating at least onesymptom of constipation-predominant irritable bowel syndrome (c-IBS) byadministering a composition, e.g., an extract or latex, from a Crotonspecies or Calophyllum species, such as a polymeric proanthocyanidincomposition. Exemplary symptoms of c-IBS include pain; abdominaldiscomfort, such as abdominal fullness, bloating or swelling; abnormalstool frequency, i.e., fewer than three bowel movements per week; hardor lumpy stools; straining during a bowel movement; urgency and feelingof incomplete bowel movement. Thus, in one embodiment, the inventionprovides a method for the treatment of one or more symptoms of c-IBScomprising administering to a patient in need of such treatment, anamount of a polymeric proanthocyanidin composition from a Croton speciesor Calophyllum species effective to treat the one or more symptoms ofc-IBS.

The present invention is based, in part, on the discovery that anextract or latex from Croton spp. or Calophyllum spp., includingpolymeric proanthocyanidin compositions isolated from Croton spp. orCalophyllum spp., including crofelemer, alleviate pain, abdominaldiscomfort and constipation associated with IBS. Additionally, thepresent invention is based, in part, on the discovery that an extract orlatex from Croton spp. or Calophyllum spp., including polymericproanthocyanidin compositions isolated from Croton spp. or Calophyllumspp., including crofelemer, alleviate symptoms of c-IBS, such asabnormal stool frequency at dosages higher than those useful to treatsymptoms of diarrhea-predominant IBS.

In one preferred embodiment, the composition of the invention is aproanthocyanidin polymer composition. In another preferred embodiment,the composition is an aqueous soluble proanthocyanidin polymercomposition. In another preferred embodiment, the compositions of thepresent invention are not substantially systemically absorbed or aremodified not to be systemically absorbed when administered orally.

Proanthocyanidins are a group of condensed tannins. Tannins are found ina wide variety of plants and are classified as either hydrolyzable orcondensed. Many plants used in traditional medicine as treatment orprophylaxis for diarrhea have been found to contain tannins andproanthocyanidins in particular (see, e.g., Yoshida et al., 1993,Phytochemistry 32:1033; Yoshida et al., 1992, Chem. Pharm. Bull.,40:1997; Tamaka et al., 1992, Chem. Pharm. Bull. 40:2092).

Proanthocyanidins are comprised of at least two or more monomer unitsthat may be of the same or different monomeric structure. The monomerunits (generally termed “leucoanthocyanidin”) are generally monomericflavonoids which include catechins, epicatechins, gallocatechins,galloepicatechins, flavanols, flavonols, and flavan-3,4-diols,leucocyanidins and anthocyanidins. Therefore, the polymer chains arebased on different structural units, which create a wide variation ofpolymeric proanthocyanidins and a large number of possible isomers(Hemingway et al., 1982, J. C. S. Perkin, 1:1217). Larger polymers ofthe flavonoid 3-ol units are predominant in most plants, and are foundwith average molecular weights above 2,000 daltons, containing 6 or moreunits (Newman et al., 1987, Mag. Res. Chem. 25:118).

Proanthocyanidin polymers are found in a wide variety of plants,particularly those with a woody habit of growth (e.g., Croton spp. andCalophyllum spp.). A number of different Croton tree species, includingCroton sakutaris, Croton gossypifolius, Croton palanostima, Crotonlechleri, Croton erythrochilus and Croton draconoides, found in SouthAmerica, produce a red viscous latex sap called Sangre de Drago or“Dragon's Blood”. This red, viscous latex is widely known for itsmedicinal properties. For example, U.S. Pat. No. 5,211,944 firstdescribed the isolation of an aqueous soluble proanthocyanidin polymercomposition from Croton spp. and the use of the composition as anantiviral agent (See also Ubillas et al., 1994, Phytomedicine, 1:77).The proanthocyanidin polymer composition was shown to have antiviralactivity against a variety of viruses including, respiratory syncytial,influenza, parainfluenza and herpes viruses. U.S. Pat. No. 5,211,944also discloses the isolation of an aqueous soluble proanthocyanidinpolymer composition from Calophyllum inophylum and the use of thiscomposition as an antiviral agent.

Exemplary proanthocyanidin polymer compositions useful in the presentinvention are preferably isolated from a Croton spp. or Calophyllum sppby any method known in the art. For example, the proanthocyanidinpolymer composition may be isolated from a Croton spp. or Calophyllumspp. by the method disclosed in Example 2, infra, or disclosed in U.S.Pat. No. 5,211,944 or in Ubillas et al., 1994, Phytomedicine 1: 77-106.

In one preferred embodiment, a proanthocyanidin polymer composition ofthe invention is crofelemer. Crofelemer (CAS 148465-45-6) is anoligomeric proanthocyanidin of varying chain lengths derived from theDragon's Blood of Croton lecheri of the family Euphorbiaceae. Crofelemerhas an average molecular weight of approximately 1900 daltons toapproximately 2700 daltons. The monomers comprising crofelemer comprisecatechin, epicatechin, gallocatechin, and epigallocatechin. The chainlength of crofelemer ranges from about 3 to about 30 units with anaverage chain length of about 8 units. The structure of crofelemer isshown below.

Another illustrative method for isolating crofelemer can be found inU.S. Patent Publication No. 2005/0019389, the contents of which areexpressly incorporated herein. PCT application PCT/US00/02687, publishedas WO 00/47062, discloses a method of manufacturing a proanthocyanidinpolymeric composition isolated from Croton spp. or Calophyllum spp.

In other embodiments of the invention, the composition useful in themethods of the invention is a raw latex obtained from a Croton speciesor a Calophyllum species or an extract obtained from a Croton species ora Calophyllum species that are not specifically polymericproanthocyanidin compositions. Exemplary extracts are described inPersinos et al., 1979, J. Pharma. Sci. 68:124 and Sethi, 1977, CanadianJ. Pharm. Sci. 12:7.

In a preferred embodiment, the compositions used in the methods of theinvention are not substantially systemically absorbed when administeredorally, i.e., only 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less than0.5% absorbed of the dosage given. Drugs that are systemically absorbedwhen delivered orally can be modified to prevent systemic absorption, ifnecessary. Such modifications are known in the art. For example, acomposition of the invention may be covalently attached to anon-systemically absorbed compound that is substantially inert in thegastrointestinal tract and does not interfere with the function of thecomposition. Such non-systemically absorbed compounds include variouspolymers. The polymers that are preferably used with the compositions ofthe invention resist degradation and absorption in the gastrointestinalsystem, i.e., the polymers do not substantially break down underphysiological conditions in the stomach and intestines into fragmentswhich are absorbable by body tissues. Polymers that have anon-hydrolyzable backbone which is substantially inert under conditionsencountered in the gastrointestinal tract, are preferred. Such polymerswill preferably have a sufficiently high molecular weight to resistabsorption, partially or completely, from the gastrointestinal tractinto other parts of the body. The polymers can have molecular weightsranging from about 500 Daltons to about 500,000 Daltons, preferably fromabout 2,000 Daltons to about 150,000 Daltons. Examples of suitablepolymers include but are not limited to, polysaccharides, polyethyleneglycol polymers, cellulosic polymers, polystyrene polymers, polyacrylatepolymers, and polyamide polymers.

The present invention encompasses methods for treating and/or preventingone or more symptoms associated with constipation-predominant irritablebowel syndrome (c-IBS), in warm blooded animals, including male andfemale humans, which symptoms include, but are not limited to, pain,abdominal discomfort and abnormal stool frequency. The methods of theinvention generally comprise administering to a subject in need of c-IBStreatment a polymeric proanthocyanidin composition in accordance withthe invention. In a preferred embodiment, the composition is orallyadministered and is not systemically absorbed. In another embodiment,the composition is enteric protected. Preferably, the patient is a humanfemale.

In one embodiment, the present invention provides a method of treatingpain associated with c-IBS comprising administering to a patient in needof such treatment, an amount of a polymeric proanthocyanidin compositioneffective to treat pain associated with c-IBS, in which said amount isbioequivalent to an orally administered dose of about 250 mg per day toabout 4000 mg per day of crofelemer. In another embodiment, the presentinvention provides a method of treating abdominal discomfort associatedwith c-IBS comprising administering to a patient in need of suchtreatment, an amount of a polymeric proanthocyanidin compositioneffective to treat abdominal discomfort associated with c-IBS, in whichsaid amount is bioequivalent to an orally administered dose of about 250mg per day to about 4000 mg per day of crofelemer. In certainembodiments, the composition is co-administered with an analgesic and/oranti-inflammatory compound.

Pain and discomfort can be measured by any method known in the art, forinstance on a pain or discomfort scale in which a patient assigns thelevel of pain or discomfort on a scale of 0 to 5, with 0 being no painor discomfort and 5 being assigned the highest level of pain ordiscomfort. In certain embodiments, the alleviation of pain ordiscomfort is measured by a lowering of the average level of pain ordiscomfort, an increase in the number of pain or discomfort-free days.In certain embodiments, the number of pain or discomfort-free days isincreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or byat least 50% compared to before treatment. In other embodiments, thelevel of pain or discomfort decreased by at least 0.1, 0.2, 0.3, 0.4 orby at least 0.5 units compared to before treatment, or even by at least1 unit, 1.5 units, or 2 units.

In another embodiment, the present invention provides a method oftreating abnormal stool frequency associated with c-IBS comprisingadministering to a patient in need of such treatment, an amount of apolymeric proanthocyanidin composition effective to treat the abnormalstool frequency associated with c-IBS.

In particular embodiments, stool frequency is increased by at least 10%,20%, 30%, 40% or 50% or more compared to before treatment. In otherembodiments, stool frequency is increased by at least one bowel movementper day compared to before treatment. In yet another embodiment, stoolfrequency is increased by at least one, two, three or four additionalbowel movements per week compared to before treatment. In certainembodiments, stool consistency is also decreased, i.e., there is anincrease in the amount of water in the stool, by at least 10%, 20%, 25%,30%, 40%, or 50% compared to before treatment.

In an alternative embodiment, the present invention encompasses methodsfor treating and/or preventing one or more symptoms associated withalternating diarrhea/constipation-predominant irritable bowel syndrome(a-IBS), in warm blooded animals, including male and female humans,which symptoms include, but are not limited to, pain, abdominaldiscomfort, abnormal stool frequency, abnormal stool consistency,diarrhea, and constipation. The methods of the invention generallycomprise administering to a subject in need of a-IBS treatment apolymeric proanthocyanidin composition in accordance with the invention.In a preferred embodiment, the composition is orally administered and isnot systemically absorbed. In another embodiment, the composition isenteric protected. Preferably, the patient is a human female.

In one embodiment, the present invention provides a method of treatingpain associated with a-IBS comprising administering to a patient in needof such treatment, an amount of a polymeric proanthocyanidin compositioneffective to treat pain associated with a-IBS. In another embodiment,the present invention provides a method of treating abdominal discomfortassociated with a-IBS comprising administering to a patient in need ofsuch treatment, an amount of a polymeric proanthocyanidin compositioneffective to treat abdominal discomfort associated with a-IBS. Incertain embodiments, the composition is co-administered with ananalgesic and/or anti-inflammatory compound. Pain and discomfort can bemeasured by any method known in the art, for instance on a pain ordiscomfort scale in which a patient assigns the level of pain ordiscomfort on a scale of 0 to 5, with 0 being no pain or discomfort and5 being assigned the highest level of pain or discomfort. In certainembodiments, the alleviation of pain or discomfort is measured by alowering of the average level of pain or discomfort, an increase in thenumber of pain- or discomfort-free days. In certain embodiments, thenumber of pain- or discomfort-free days is increased by at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or by at least 50% compared tobefore treatment. In other embodiments, the level of pain or discomfortdecreased by at least 0.1, 0.2, 0.3, 0.4 or by at least 0.5 unitscompared to before treatment.

In another embodiment, the present invention provides a method oftreating constipation associated with a-IBS comprising administering toa patient in need of such treatment, an amount of a polymericproanthocyanidin composition effective to treat the constipationassociated with a-IBS.

In particular embodiments, stool frequency is increased by at least 10%,20%, 30%, 40% or 50% or more compared to before treatment. In otherembodiments, stool frequency is increased by at least one bowel movementper day compared to before treatment. In yet another embodiment, stoolfrequency is increased by at least one, two, three or four additionalbowel movements per week compared to before treatment. In certainembodiments, stool consistency is also decreased, i.e., there is anincrease in the amount of water in the stool, by at least 10%, 20%, 25%,30%, 40%, or 50% compared to before treatment.

In another embodiment, the present invention provides a method oftreating diarrhea associated with a-IBS comprising administering to apatient in need of such treatment, an amount of a polymericproanthocyanidin composition isolated from Croton spp. or Calophyllumspp. effective to treat the diarrhea associated with a-IBS, in whichsaid amount is bioequivalent to an orally administered dose of about 50mg per day to about 4000 mg per day of crofelemer. In yet anotherembodiment, the present invention provides a method of treating abnormalstool frequency, abnormal stool consistency or presence of urgencyassociated with a-IBS comprising administering to a patient in need ofsuch treatment, an amount of a polymeric proanthocyanidin compositionisolated from Croton spp. or Calophyllum spp. effective to treat theabnormal stool frequency, abnormal stool consistency or presence ofurgency associated with a-IBS, in which said amount is bioequivalent toan orally administered dose of about 50 mg per day to about 4000 mg perday of crofelemer. In particular embodiments, stool frequency isdecreased by at least 10%, 20%, 30%, 40% or 50% compared to beforetreatment. In other embodiments, stool frequency is decreased by atleast one bowel movement per day compared to before treatment. In otherembodiments, stool consistency is increased, i.e., there is a decreasein the amount of water in the stool, by at least 10%, 20%, 25%, 30%,40%, or 50% compared to before treatment. In yet other embodiments,presence of urgency is decreased by at least 10%, 20%, 30%, 40%, or byat least 50% compared to before treatment.

The compositions of the invention can be administered in a single or adivided dosage from one, two, three or four times per day. In aparticular embodiment, the composition is administered twice daily. Inyet another embodiment, the composition is administered twice daily forat least two consecutive days. In yet another embodiment, thecomposition is administered for at least a period of time selected fromthe group consisting of 24 hours, 48 hours, 72 hours, 96 hours, oneweek, two weeks, one month, two months, and three months. In certainembodiments, where c-IBS or a-IBS is a chronic condition, thecomposition is taken indefinitely.

The polymeric proanthocyanidin compositions of the present invention arepreferably administered orally. In certain embodiments, however, methodsof administering a composition of the invention include, but are notlimited to, parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), and mucosal (e.g.,intranasal route). In other embodiments, a composition is administeredintramuscularly, intravenously, or subcutaneously. Compositions may beadministered by any convenient route, for example, by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local.

In certain preferred embodiments of the present invention, the polymericproanthocyanidin composition is crofelemer (CAS 148465-45-6). In otherpreferred aspects, crofelemer is administered orally. In yet otherpreferred embodiments, the composition is formulated so as to protectthe composition from the stomach environment, i.e., from the acidicenvironment and digestive proteins found in the stomach. In a preferredembodiment, administration is by oral route and the composition isenteric protected crofelemer.

In certain preferred embodiments, crofelemer is orally administered inan enteric protected form (enteric coated) in a total amount of not lessthan about 250 mg/day. As used herein, about means within the margin oferror. In specific embodiments, the enteric coated crofelemer is orallyadministered to a subject suffering from c-IBS in an amount of fromabout 250 mg/day to 4000 mg/day. In other specific embodiments, wherethe subject is suffering from a-IBS, the enteric coated crofelemer isorally administered in an amount of from about 50 mg/day to 4000 mg/day.In another embodiment, the enteric coated crofelemer is orallyadministered to a subject in a total amount of not less than about 500mg/day. In specific embodiments, the enteric coated crofelemer is orallyadministered to a subject in an amount of from about 250 mg/day to about2000 mg/day. In other embodiments, the enteric coated crofelemer isorally administered to a subject at not less than about 1750 mg/day,about 1700 mg/day, about 1650 mg/day, about 1600 mg/day, about 1550mg/day, about 1500 mg/day, about 1450 mg/day, about 1400 mg/day, about1350 mg/day, about 1300 mg/day, about 1250 mg/day, 1200 mg/day, about1150 mg/day, about 1100 mg/day, about 1050 mg/day, about 1000 mg/day,about 950 mg/day, about 900 mg/day, about 850 mg/day, about 800 mg/day,about 750 mg/day, about 700 mg/day, 650 mg/day, about 600 mg/day, about550 mg/day, about 500 mg/day, about 450 mg/day, about 400 mg/day, about350 mg/day, about 300 mg/day, or about 250 mg/day of orally administeredenteric coated crofelemer. In yet another embodiment, the enteric coatedcrofelemer is orally administered to a subject in an amount from about500 mg/day to 1000 mg/day. In other embodiments, the enteric coatedcrofelemer is orally administered to a subject in an amount of fromabout 250 mg/day to about 2000 mg/day, from about 250 mg/day to about1000 mg/day, from about 250 mg/day to about 750 mg/day, from about 250mg/day to about 500 mg/day, from about 350 mg/day to about 650 mg/day,or from about 500 mg/day to about 750 mg/day. In other particularembodiments, the total dosage of the enteric coated crofelemer orallyadministered to a subject is not less than about 250 mg, about 255 mg,about 260 mg, about 265 mg, about 285 mg, about 290 mg, about 295 mg,about 315 mg, about 320 mg, about 325 mg, about 345 mg, about 350 mg,about 355 mg, about 375 mg, about 380 mg, about 385 mg, about 405 mg,about 410 mg, about 415 mg, about 435 mg, about 440 mg, about 445 mg,about 465 mg, about 470 mg, about 475 mg, about 495 mg, about 500 mg,about 505 mg, about 525 mg, about 530 mg, about 535 mg, about 555 mg,about 560 mg, about 565 mg, about 585 mg, about 590 mg, about 595 mg,about 615 mg, about 620 mg, about 625 mg, about 645 mg, about 650 mg,about 655 mg, about 675 mg, about 680 mg, about 685 mg, about 705 mg,about 710 mg, about 715 mg, about 270 mg, about 275 mg, about 280 mg,about 300 mg, about 305 mg, about 310 mg, about 330 mg, about 335 mg,about 340 mg, about 360 mg, about 365 mg, about 370 mg, about 390 mg,about 395 mg, about 400 mg, about 420 mg, about 425 mg, about 430 mg,about 450 mg, about 455 mg, about 460 mg, about 480 mg, about 485 mg,about 490 mg, about 510 mg, about 515 mg, about 520 mg, about 540 mg,about 545 mg, about 550 mg, about 570 mg, about 575 mg, about 580 mg,about 600 mg, about 605 mg, about 610 mg, about 630 mg, about 635 mg,about 640 mg, about 660 mg, about 665 mg, about 670 mg, about 690 mg,about 695 mg, about 700 mg, about 720 mg, about 725 mg, about 730 mg,about 735 mg, about 740 mg, about 745 mg, about 750 mg, about 755 mg,about 760 mg, about 765 mg, about 770 mg, about 775 mg, about 780 mg,about 785 mg, about 790 mg, about 795 mg, about 800 mg, about 805 mg,about 810 mg, about 815 mg, about 820 mg, about 825 mg, about 830 mg,about 835 mg, about 840 mg, about 845 mg, about 850 mg, about 855 mg,about 860 mg, about 865 mg, about 870 mg, about 875 mg, about 880 mg,about 885 mg, about 890 mg, about 895 mg, about 900 mg, about 905 mg,about 910 mg, about 915 mg, about 920 mg, about 925 mg, about 930 mg,about 935 mg, about 940 mg, about 945 mg, about 950 mg, about 955 mg,about 960 mg, about 965 mg, about 970 mg, about 975 mg, about 980 mg,about 985 mg, about 990 mg, about 995 mg, or not less than about 1000mg, once, twice, or three-times per day.

In other embodiments of the invention, the composition, theproanthocyanidin polymer composition is preferably given at a dosagethat is bioequivalent to orally administered enteric coated crofelemerat a dosage of about 250 mg per day to about 4000 mg/day or any of thedoses listed above.

In a preferred embodiment, crofelemer is enteric coated so as to protectit from degradation by the acidic conditions of the stomach and/or frominteractions with proteins, such as pepsin, present in the stomach,i.e., an enteric protected formulation. In a specific embodiment,crofelemer is in tablet form. In yet another specific embodiment, thetablet is enteric coated, e.g., EUDRAGIT®. In a preferred embodiment,crofelemer is formulated as an enteric coated bead or granule in anenteric coated capsule shell. In another embodiment, crofelemer isformulated in a delayed release composition, e.g., Merck GEM, Alza OROS,wax matrix (release is primarily delayed until after the formulationpasses out of the stomach and into the intestine).

In certain embodiments, the composition is formulated with a compound orcompounds which neutralize stomach acid. Alternatively, thepharmaceutical composition containing the composition is administeredeither concurrent with or subsequent to or after administration of apharmaceutical composition which neutralize stomach acid. Compounds,such as antacids, which are useful for neutralizing stomach acidinclude, but are not limited to, aluminum carbonate, aluminum hydroxide,bismuth subnitrate, bismuth subsalicylate, calcium carbonate,dihydroxyaluminum sodium carbonate, magaldrate, magnesium carbonate,magnesium hydroxide, magnesium oxide, and mixtures thereof. Compoundsthat are able to reduce the secretion of stomach acid and/or are able toreduce the acidity of stomach fluid are well known in the art andinclude, but are not limited to, antacids (aluminum hydroxide, aluminumcarbonate, aluminum glycinate, magnesium oxide, magnesium hydroxide,magnesium carbonate, calcium carbonate, sodium bicarbonate), stomachacid blockers (cimetidine (Tagamet™), famotidine (Mylanta™, Pepcid™),nizatidine (Axid™), ranitidine (Zantac™), omeprazole (Zegerid™)) and acombination of any of the foregoing. In general, any drug that has beenapproved for sale by the relevant government agency and is able toreduce the production of stomach acid and/or reduce the acidity ofstomach fluid can be administered in combination with an inhibitormolecule, such as crofelemer, in accordance with the methods of theinvention.

In other embodiments, the composition is administered with othercompounds which are useful in treating constipation or pain, whichcompounds are preferably not systemically absorbed. Such compoundsinclude 5-ASA, sulfasalazine, mesalamine, APAZA, celecoxib androfecoxib.

In a particular embodiment where crofelemer is not enteric coated,crofelemer is formulated with one or more compounds that are able toreduce the secretion of stomach acid and/or able to reduce the acidityof stomach fluid. Preferably, the dosage of crofelemer to be given inthis formulation is a dosage that is bioequivalent to orallyadministered enteric coated crofelemer at a dosage of about 250 mg perday to about 4000 mg per day. In an exemplary embodiment, crofelemer isformulated in a controlled release (delayed release) composition.

In other embodiments, the compositions of the invention can beadministered in combination with analgesic or anti-inflammatory agents.In a preferred embodiment, the analgesic or anti-inflammatory agent isformulated or modified such that it is not substantially systemicallyabsorbed, i.e., only 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.5% absorbedof the dosage given.

The present invention also provides pharmaceutical formulations of thepolymeric proanthocyanidin compositions of the invention comprising thecomposition along with a pharmaceutically acceptable vehicle, at a dosewhich is therapeutically effective at treating and/or ameliorating oneor more symptoms associated with c-IBS or a-IBS. In one embodiment, adirectly compressible proanthocyanidin polymer composition (i.e., thatcan be directly compressed, without excipients, into a tablet ofpharmaceutically acceptable hardness and friability) compressed into atablet, optionally with a lubricant, such as but not limited tomagnesium stearate, is enteric coated. In another embodiment, thepharmaceutical compositions of the invention alternatively include oneor more substances that either neutralize stomach acid and/or enzymes orare active to prevent secretion of stomach acid. These formulations canbe prepared by methods known in the art, see, e.g., methods described inRemington's Pharmaceutical Sciences, 18th Ed., ed. Alfonso R. Gennaro,Mack Publishing Co., Easton, Pa., 1990.

In another preferred embodiment, the pharmaceutical compositioncomprises a proanthocyanidin polymer composition prepared from a Crotonspp, the dosage of which is not less than a bioequivalent dosage oforally administered enteric protected crofelemer of 250 mg per day,preferably not less than 500 mg/day. In a preferred embodiment, theproanthocyanidin polymer composition of the present invention iscrofelemer (CAS 148465-45-6).

The compositions of the invention can be provided in any therapeuticallyacceptable pharmaceutical form. The pharmaceutical composition can beformulated for oral administration as, for example but not limited to,drug powders, crystals, granules, small particles (which includeparticles sized on the order of micrometers, such as microspheres andmicrocapsules), particles (which include particles sized on the order ofmillimeters), beads, microbeads, pellets, pills, microtablets,compressed tablets or tablet triturates, molded tablets or tablettriturates, and in capsules, which are either hard or soft and containthe composition as a powder, particle, bead, solution or suspension. Thepharmaceutical composition can also be formulated for oraladministration as a solution or suspension in an aqueous liquid, as aliquid incorporated into a gel capsule or as any other convenientformulation for administration, or for rectal administration, as asuppository, enema or other convenient form. The compositions of theinvention can also be provided as a controlled release system (see,e.g., Langer, 1990, Science 249: 1527-1533).

The pharmaceutical formulation can also include any type ofpharmaceutically acceptable excipients, additives or vehicles. Forexample, but not by way of limitation, diluents or fillers, such asdextrates, dicalcium phosphate, calcium sulfate, lactose, cellulose,kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose,inositol, powdered sugar, bentonite, microcrystalline cellulose, orhydroxypropylmethylcellulose may be added to the inhibitor molecule toincrease the bulk of the composition. Also, binders, such as but notlimited to, starch, gelatin, sucrose, glucose, dextrose, molasses,lactose, acacia gum, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapgol husks, carboxymethylcellulose,methylcellulose, polyvinylpyrrolidone, Veegum and starch arabogalactan,polyethylene glycol, ethylcellulose, and waxes, may be added to theformulation to increase its cohesive qualities. Additionally,lubricants, such as but not limited to, talc, magnesium stearate,calcium stearate, stearic acid, hydrogenated vegetable oils,polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,leucine, carbowax, sodium lauryl sulfate, and magnesium lauryl sulfatemay be added to the formulation. Also, glidants, such as but not limitedto, colloidal silicon dioxide or talc may be added to improve the flowcharacteristics of a powdered formulation. Finally, disintegrants, suchas but not limited to, starches, clays, celluloses, algins, gums,crosslinked polymers (e.g., croscarmelose, crospovidone, and sodiumstarch glycolate), Veegum, methylcellulose, agar, bentonite, celluloseand wood products, natural sponge, cation-exchange resins, alginic acid,guar gum, citrus pulp, carboxymethylcellulose, or sodium lauryl sulfatewith starch may also be added to facilitate disintegration of theformulation in the intestine.

In one aspect of this embodiment, crofelemer is formulated for oraladministration. In other aspects, the pharmaceutical dosage form isformulated to protect the composition, e.g., crofelemer, fromdegradation by the acidic conditions of the stomach and frominteractions with proteins, such as pepsin, present in the stomach.Thus, in a preferred aspect, the formulation is enteric coated. Forexample, the enteric coated formulation is enteric coated tablets, beadsor granules, which optionally contain a lubricant such as, but notlimited to, magnesium stearate. The enteric coated formulations includeenteric coated beads in a capsule, enteric coated microspheres in acapsule, enteric coated microspheres provided in a suspension or mixedwith food, which suspensions are particularly convenient for pediatricadministration, and enteric coated compressed tablets. The capsule canbe a hard-shell gelatin capsule or a cellulose capsule. In particular,the pharmaceutical composition is formulated as an enteric coatedcapsule. In one specific aspect, a proanthocyanidin polymer compositionis administered in tablet form, which tablet is backfilled withmicrocrystalline cellulose.

In one embodiment, the composition is directly compressed, that is, thecomposition of the invention, with or without any excipients, can becompressed into a tablet, or other pharmaceutical formulation, that hasa pharmaceutically acceptable hardness and friability. Preferably, thedirectly compressible pharmaceutical composition can be compressed intotablets having a hardness of greater than 4 kp (kiloponds), preferably ahardness of 8 to 14 kp, more preferably a hardness of 10 to 13 kp. Adirectly compressible composition can be compressed into a tablet thathas a friability of not more than 1% loss in weight, preferably lessthan 0.8% loss in weight, more preferably less than 0.5% loss in weight.

In a preferred embodiment, the directly compressible formulationconsists of 99.93% crofelemer and 0.07% magnesium stearate and is coatedwith a methacrylic acid copolymer. In another preferred embodiment, thepharmaceutical formulation contains a directly compressible compositionbut no excipients, additives or vehicles other than an enteric coating;however, the formulation may contain a lubricant, such as but notlimited to, magnesium stearate. Preferably, a directly compressedproanthocyanidin polymer composition formulation is formulated as atablet of pharmaceutically acceptable hardness (greater than 4 kp,preferably 8-14 kp, and more preferably 10-13 kp) and friability (notmore than 1% loss in weight, preferably less than 0.8% loss in weight,and more preferably less than 0.5% loss in weight).

In a more preferred embodiment, a composition of the invention isenteric coated. Enteric coatings are those coatings that remain intactin the stomach, but will dissolve and release the contents of the dosageform once it reaches the small intestine. A large number of entericcoatings are prepared with ingredients that have acidic groups suchthat, at the very low pH present in the stomach, i.e. pH 1.5 to 2.5, theacidic groups are not ionized and the coating remains in anundissociated, insoluble form. At higher pH levels, such as in theenvironment of the intestine, the enteric coating is converted to anionized form, which can be dissolved to release the inhibitor molecule.Other enteric coatings remain intact until they are degraded by enzymesin the small intestine, and others break apart after a defined exposureto moisture, such that the coatings remain intact until after passageinto the small intestines.

Polymers which are useful for the preparation of enteric coatingsinclude, but are not limited to, shellac, starch and amylose acetatephthalates, styrene-maleic acid copolymers, cellulose acetate succinate,cellulose acetate phthalate (CAP), polyvinylacetate phthalate (PVAP),hydroxypropylmethylcellulose phthalate (grades HP-50 and HP-55),ethylcellulose, fats, butyl stearate, and methacrylic acid-methacrylicacid ester copolymers with acid ionizable groups (including “ACRYLEZE®”and “EUDRAGIT®”), such as “EUDRAGIT® L 30D”, “EUDRAGIT® RL 30D”,“EUDRAGIT® RS 30D”, “EUDRAGIT® L 100-55”, and “EUDRAGIT® L 30D-55”. In apreferred embodiment, the pharmaceutical composition contains apolymeric proanthocyanidin composition and the enteric coating polymer“EUDRAGIT® L 30D”, an anionic copolymer of methacrylic acid and methylacrylate with a mean molecular weight of 250,000 Daltons. In anotherpreferred embodiment, the enteric coating polymer is “EUDRAGIT® L30D-55”.

The disintegration of the enteric coating occurs either by hydrolysis byintestinal enzymes or by emulsification and dispersion by bile salts,depending upon the type of coating used. For example, esteraseshydrolyze esterbutyl stearate to butanol and stearic acid and, as thebutanol dissolves, the stearic acid flakes off of the medicament.Additionally, bile salts emulsify and disperse ethylcellulose,hydroxypropylmethylcellulose, fats and fatty derivatives. Other types ofcoatings are removed depending on the time of contact with moisture, forexample coatings prepared from powdered carnauba wax, stearic acid, andvegetable fibers of agar and elm bark rupture after the vegetable fibersabsorb moisture and swell. The time required for disintegration dependsupon the thickness of the coating and the ratio of vegetable fibers towax.

Application of the enteric coating to the polymeric proanthocyanidincomposition of the invention can be accomplished by any method known inthe art for applying enteric coatings. For example, but not by way oflimitation, the enteric polymers can be applied using organic solventbased solutions containing from 5 to 10% w/w polymer for sprayapplications and up to 30% w/w polymer for pan coatings. Solvents thatare commonly in use include, but are not limited to, acetone,acetone/ethyl acetate mixtures, methylene chloride/methanol mixtures,and tertiary mixtures containing these solvents. Some enteric polymers,such as methacrylic acid-methacrylic acid ester copolymers can beapplied using water as a dispersant. The volatility of the solventsystem must be tailored to prevent sticking due to tackiness and toprevent high porosity of the coating due to premature spray drying orprecipitation of the polymer as the solvent evaporates.

Furthermore, plasticizers can be added to the enteric coating to preventcracking of the coating film. Suitable plasticizers include the lowmolecular weight phthalate esters, such as diethyl phthalate, acetylatedmonoglycerides, triethyl citrate, polyethyl glycoltributyl citrate andtriacetin. Generally, plasticizers are added at a concentration of 10%by weight of enteric coating polymer weight. Other additives such asemulsifiers, for example detergents and simethicone, and powders, forexample talc, may be added to the coating to improve the strength andsmoothness of the coating. Additionally, pigments may be added to thecoating to add color to the pharmaceutical formulation.

In preferred embodiments, a pharmaceutical composition of the polymericproanthocyanidin composition is provided as enteric coated beads inhard-shell gelatin capsules. In a preferred embodiment, proanthocyanidinpolymer beads are prepared by mixing a proanthocyanidin polymercomposition with hydroxypropylmethylcellulose and layering the mixtureonto nonpareil seeds (sugar spheres). In a more preferred embodiment,crofelemer, which is directly compressible, without any excipients,additives or vehicles other than an enteric coating, is milled andfractionated into beads (i.e., as beads that do not contain thenonpareil sugar seeds). The beads may be covered with a seal coat ofOpadry Clear (mixed with water). A preferred enteric coating of thebeads is “EUDRAGIT™ L 30D” or “EUDRAGIT™ L 30D-55” applied as an aqueousdispersion containing 20%-30% w/w dry polymer substance, which issupplied with 0.7% sodium lauryl sulfate NF (SLS) and 2.3% polysorbate80 NF (Tween™ 20) as emulsifiers, to which plasticizers, such aspolyethylene glycol and/or citric acid esters, are added to improve theelasticity of the coating, and talc can be added to reduce the tendencyof the enteric coating polymer to agglutinate during the applicationprocess and to increase the smoothness of the film coating.

In a preferred formulation, the final composition of enteric coatedproanthocyanidin polymer composition beads containing the nonpareilseeds is 17.3% w/w nonpareil seeds, 64.5% w/w proanthocyanidin polymercomposition, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadryclear, 14.5% w/w “EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate, and0.25% w/w glyceryl monostearate. This pharmaceutical formulation may beprepared by any method known in the art or by the method described inExample 1, infra.

A preferred formulation of the proanthocyanidin polymer compositionbeads not containing the nonpareil seeds is 78% w/w directlycompressible proanthocyanidin polymer composition (e.g., isolated by themethod described in the Examples), 0.76% w/w Opadry Clear, 19% w/w“EUDRAGIT™ L 30D-55”, 1.9% triethyl citrate, and 0.34% w/w glycerylmonostearate. This pharmaceutical formulation may be prepared by anymethod known in the art or by the method described in Example 2, infra.

Another preferred formulation contains 54.58% w/w proanthocyanidinpolymer composition beads (without non-pareil seeds and made of adirectly compressible proanthocyanidin polymer composition), 1.78% w/wOpadry Clear, 39% w/w “EUDRAGIT™ L 30D-55, 3.9% triethylcitrate, and0.74% w/w glyceryl monostearate.

In another embodiment, the pharmaceutical composition comprising thepolymeric proanthocyanidin composition of the invention is formulated asenteric coated granules or powder (microspheres with a diameter of300-500 g) provided in either hard shell gelatin capsules or suspendedin an oral solution for pediatric administration. The enteric coatedpowder or granules may also be mixed with food, particularly forpediatric administration. This preparation may be prepared usingtechniques well known in the art, such as the method described inExample 1 C, infra.

In general, the granules and powder can be prepared using any methodknown in the art, such as but not limited to, crystallization,spray-drying or any method of comminution, preferably using a high speedmixer/granulator. Examples of high speed mixer/granulators include the“LITTLEFORD LODIGE™” mixer, the “LITTLEFORD LODIGE™” MGTmixer/granulator, and the “GRAL™” mixer/granulator. During thehigh-shear powder mixing, solutions of granulating agents, calledbinders, are sprayed onto the powder to cause the powder particles toagglomerate, thus forming larger particles or granules. Granulatingagents which are useful for preparing the granules, include but are notlimited to, cellulose derivatives (including carboxymethylcellulose,methylcellulose, and ethylcellulose), gelatin, glucose,polyvinylpyrrolidone (PVP), starch paste, sorbitol, sucrose, dextrose,molasses, lactose, acacia gum, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks, Veegurn and larcharabogalactan, polyethylene glycol, and waxes. Granulating agents may beadded in concentrations ranging from 1 to 30% of the mass of theparticles or granules.

The powder or granules are preferably coated using the fluidized bedequipment. The granules or powder may then be covered with a seal coatof Opadry Clear (mixed with water). A preferred enteric coating is“EUDRAGIT™ L 30D” applied as an aqueous dispersion containing 30% w/wdry polymer substance, which is supplied with 0.7% sodium lauryl sulfateNF (SLS) and 2.3% polysorbate 80 NF (Tween™ 20) as emulsifiers, to whichthe plasticizers, polyethylene glycol and citric acid esters, are addedto improve the elasticity of the coating, and talc is added to reducethe tendency of the enteric coating polymer to agglutinate during theapplication process and to increase the smoothness of the film coating.In a preferred embodiment, the final composition of an enteric coatedpowder is 81.8% w/w proanthocyanidin polymer composition, 1.5% w/whydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w“EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate, and 0.25% w/w glycerylmonostearate. The final composition of the enteric coated granules is81.8% w/w proanthocyanidin polymer composition, 10%polyvinylpyrrolidone, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/wOpadry clear, 14.5% w/w “EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate,and 0.25% w/w glyceryl monostearate.

The enteric coated granules or powder particles can further be suspendedin a solution for oral administration, particularly for pediatricadministration. The suspension can be prepared from aqueous solutions towhich thickeners and protective colloids are added to increase theviscosity of the solution to prevent rapid sedimentation of the coatedpowder particles or granules. Any material which increases the strengthof the hydration layer formed around suspended particles throughmolecular interactions and which is pharmaceutically compatible with theinhibitor molecule can be used as a thickener, such as but not limitedto, gelatin, natural gums (e.g., tragacanth, xanthan, guar, acacia,panwar, ghatti, etc.), and cellulose derivatives (e.g., sodiumcarboxymethylcellulose, hydroxypropylcellulose, andhydroxypropylmethylcellulose, etc.). Optionally, a surfactant such asTween™ may be added to improve the action of the thickening agent. Apreferred suspension solution is a 2% w/w hydroxypropylmethylcellulosesolution in water containing 0.2% Tween™

The polymeric proantocyanidin composition can also be formulated asenteric coated tablets. In one preferred embodiment, a proanthocyanidinpolymer composition is granulated with any pharmaceutically acceptablediluent (such as those listed above) by the methods described above forpreparing the granules. Then, the granules are compressed into tabletsusing any method well known in the art, for example but not limited to,the wet granulation method, the dry granulation method or the directcompression method. Preferred diluents include, but are not limited to,microcrystalline cellulose (“AVICEL™ PH 200/300”) and dextrates(“EMDEX™”). Additionally, disintegrants, such as those described above,and lubricants, such those described above, may also be added to thetablet formulation. A preferred tablet formulation contains 250 mgproanthocyanidin polymer composition, 7 mg of the disintegrant“AC-DI-SOL™” (cross linked sodium carboxymethylcellulose), 1.75 mg ofthe lubricant magnesium stearate and the weight of “AVICEL™ PH 200/300”necessary to bring the mixture up to 350 mg. The tablets are coated withan enteric coating mixture prepared from 250 grams “EUDRAGIT™ L 30 D-55,7.5 grams triethyl citrate, 37.5 grains talc and 205 grams water. Thisformulation may be prepared by any method well known in the art.

In a preferred embodiment, a directly compressible proanthocyanidinpolymer composition is made into granules by size reduction (e.g., asdescribed above) and mixed with a lubricant, preferably, magnesiumstearate. Then, the lubricated granules are compressed into tabletsusing any method well-known in the art, for example but not limited to,the direct compression method. Preferably, each tablet is 125 mgcontaining 99.6% w/w directly compressible proanthocyanidin polymercomposition and 0.40% w/w magnesium stearate. The tablets are thenpreferably coated with an enteric coating mixture of a 30% suspension(6.66 g in 22.22 g) of “EUDRAGIT™ L 30D-55”, 0.67 g triethyl citrate,1.67 g talc and 20.44 g purified water, per 100 grams of tablet. Thetablets can be prepared by any method known in the art or by the methoddescribed in Example 1E, infra.

In another embodiment, a directly compressible proanthocyanidin polymercomposition is formulated into core tablets of either 125 mg, 250 mg or500 mg containing 99.6% w/w directly compressible proanthocyanidinpolymer composition and 0.40% w/w magnesium stearate. The tablets arethen preferably coated with an enteric coating mixture. The finalcomposition of the tablets is 86.6% w/w directly compressibleproanthocyanidin polymer composition, 0.4% magnesium stearate, 6.5%“EUDRAGIT™ L30D-55”, 0.9% triethyl citrate, 2.87% talc, and 2.74% whitedispersion. The tablets can be prepared by any method known in the art,for example but not limited to the method described infra.

The compositions formed into small particles (which include particlessized on the order of micrometers, such as micro spheres andmicrocapsules), particles (which include particles sized on the order ofmillimeters), drug crystals, pellets, pills and microbeads can be coatedusing a fluidized-bed process. This process uses fluidized-bedequipment, such as that supplied by “GLATT™”, “AEROMATIC™”, “WURSTER™”,or others, by which the composition cores are whirled up in a closedcylindrical vessel by a stream of air, introduced from below, and theenteric coat is formed by spray drying it onto the cores during thefluidization time. To coat tablets or capsules, Accela-Cota coatingequipment (“MANESTY™”) can be used. By this process, the tablets orcapsules are placed in a rotating cylindrical coating pan with aperforated jacket and spraying units are installed within the pan andthe dry air is drawn in through the rotating tablets or capsules. Anyother type of coating pan, such as the “COMPU-LAB™” pan, Hi-coates“GLATT™” immersion sword process, the “DRIAM™” Dricoater, “STEINBERG™”equipment, “PELLEGRINI™” equipment, or “WALTHER™” equipment can also beused.

The pharmaceutical formulations of the invention can also be used totreat c-IBS in non-human animals, particularly in farm animals, such asbut not limited to, bovine animals, swine, ovine animals, poultry (suchas chickens), and equine animals, and other domesticated animals such ascanine animals and feline animals. In particular, the pharmaceuticalformulations of the invention can be used to treat c-IBS disease innon-human animals, particularly food animals such as cattle, sheep andswine by incorporating the pharmaceutical compositions of the inventioninto the animal's feed.

According to the methods of the present invention, the pharmaceuticalcompositions of comprising polymeric proanthocyanidin compositions ofthe invention are administered to a subject in a total amount that isbioequivalent to not less than 50 mg/day of orally administered entericprotected crofelemer. In specific embodiments, pharmaceuticalcompositions comprising crofelemer are administered to a subject in anamount of between about 50 mg per day and about 4000 mg/day.

In determining whether a subject has constipation-predominant IBS oralternating diarrhea/constipation predominant-IBS, any method can beused in the art to diagnose the subject including, but not limited to,the Rome II criteria for diagnosis of irritable bowel syndrome (Thompsonet al., 1999, Gut 45 (Suppl II):I1-43-1147). Briefly, the Rome IIdiagnostic criteria state that for at least 12 weeks, which need not beconsecutive, in the preceding 12 months of abdominal discomfort or painthat has two of three features: (1) relief with defecation, and/or (2)onset associated with a change in frequency of stool; and/or (3) onsetassociated with a change in form (appearance) of stool. Exemplarysymptoms of c-IBS include pain; abdominal discomfort, such as abdominalfullness, bloating or swelling; abnormal stool frequency, i.e., fewerthan three bowel movements per week; hard or lumpy stools; strainingduring a bowel movement; urgency and feeling of incomplete bowelmovement. For example, abnormal stool frequency, e.g., fewer than threetimes a week, supports the diagnosis of c-IBS. Exemplary symptoms ofa-IBS include periods of fewer than three bowel movements per week;periods of more that three bowel movements pre day; sometimes hard orlumpy stools, sometimes loose or watery stools; straining during a bowelmovement; urgency; feeling of incomplete bowel movement; passing mucusduring a bowel movement and abdominal fullness, bloating and swelling.Patients who switch between constipation and diarrhea have consistentabdominal pain and discomfort with both.

DEFINITIONS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. The practice of the present invention willemploy, unless otherwise indicated, conventional techniques of molecularbiology (including recombinant techniques), microbiology, cell biology,biochemistry, nucleic acid chemistry, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Current Protocols in Immunology (J. E. Coligan etal., eds., 1999, including supplements through 2001); Current Protocolsin Molecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); Molecular Cloning: A Laboratory Manual, thirdedition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction,(Mullis et al., eds., 1994); The Immunoassay Handbook (D. Wild, ed.,Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson,ed., Academic Press, 1996); Methods of Immunological Analysis (R.Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlagsgesellschaft mbH, 1993), Harlow and Lane Using Antibodies: A LaboratoryManual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1999; and Beaucage et al., eds., Current Protocols in Nucleic AcidChemistry John Wiley & Sons, Inc., New York, 2000).

As used herein, the term “derivative” in the context of anon-proteinaceous derivative refers to a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl, amine group, esterification,alkylation or phosphorylation, immobilization or addition of a polymer.

As used herein, the term “polymer” refers to compounds comprising threeor more monomeric units which may be the same or different. Thus,“polymer” refers to high molecular weight and/or insoluble polymers aswell as low molecular weight and/or soluble oligomers.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., amonkey, such as a cynomolgous monkey, and a human), and more preferablya human. In a preferred embodiment, the subject is a human. In a oneembodiment, the term “subject” excludes those subjects who suffer fromor have been diagnosed with secretory (acute) diarrhea.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe prevention, reduction, amelioration or elimination of a symptom orcomplication of c-IBS.

The term “prevention, reduction, amelioration or elimination of asymptom of c-IBS” in the context of the present invention refers to atleast one of the following: prevention of c-IBS before it occurs, forexample, in patients that suffered in the past from c-IBS but are now ina period of remission; elimination of established c-IBS (as determinedby, for example, the return of normal stool frequency); elimination ofpain associated with c-IBS; reduction of an undesired symptom of thedisease as manifested by a decrease in the severity of an existingcondition of c-IBS; elimination or reduction of one or more medicationsused in treating the subject. The reduction in the undesired symptom maybe determined by, for example, measuring stool frequency, determiningstool consistency, determining pain associated with c-IBS. Any amount ofreduction in the severity of a symptom, even if some of the symptomremains at a lower, more acceptable level (“management”), is encompassedby the term herein defined. Such remediation may be evident as anincrease in stool frequency, lessening of pain.

Since c-IBS is always accompanied by other non diarrhea-relatedsymptoms, such as abdominal discomfort, pain, bloating, fatigue, sleepdisturbances, sexual dysfunction, headache, fibromyalgia (muscleaching), dyspepsia (upper abdominal discomfort or pain), chest pain,urinary or gynecological symptoms, anxiety and depression. Reduction inat least one of these symptoms is also encompassed by the term“prevention reduction, management or elimination of a symptom orcomplication of c-IBS.”

As used herein, the terms “treat”, “treatment” and “treating” refer tothe prevention, reduction, amelioration or elimination of a symptom orcomplication of a-IBS.

The term “prevention, reduction, amelioration or elimination of asymptom of a-IBS” in the context of the present invention refers to atleast one of the following: prevention of a-IBS before it occurs, forexample, in patients that suffered in the past from a-IBS but are now ina period of remission; elimination of established a-IBS (as determinedby, for example, the return of normal stool frequency); elimination ofpain associated with a-IBS; reduction of an undesired symptom of thedisease as manifested by a decrease in the severity of an existingcondition of a-IBS; elimination or reduction of one or more medicationsused in treating the subject. The reduction in the undesired symptom maybe determined by, for example, measuring stool frequency, determiningstool consistency, determining pain associated with a-IBS. Any amount ofreduction in the severity of a symptom, even if some of the symptomremains at a lower, more acceptable level (“management”), is encompassedby the term herein defined. Such remediation may be evident as anincrease in stool frequency, lessening of pain.

Since a-IBS is always accompanied by other nondiarrhea/constipation-related symptoms, such as abdominal discomfort,pain, bloating, fatigue, sleep disturbances, sexual dysfunction,headache, fibromyalgia (muscle aching), dyspepsia (upper abdominaldiscomfort or pain), chest pain, urinary or gynecological symptoms,anxiety and depression. Reduction in at least one of these symptoms isalso encompassed by the term “prevention reduction, management orelimination of a symptom or complication of a-IBS.”

As used herein, the term “therapeutically effective amount” refers tothat amount of the therapeutic agent sufficient to result in thetreatment of c-IBS or a-IBS, to prevent advancement of c-IBS or a-IBS,cause regression of c-IBS or a-IBS, or to enhance or improve thetherapeutic effect(s) of another therapeutic agent administered to treator prevent c-IBS or a-IBS.

The following series of Examples are presented for purposes ofillustration and not by way of limitation on the scope of the invention.

EXAMPLES Example 1 Preparation of Pharmaceutical Formulations

Described below are illustrative methods for the manufacture andpackaging for different preferred pharmaceutical formulations of theproanthocyanidin polymer composition from C. lechleri according to thepresent invention.

1A. Encapsulated Enteric Coated Beads Detailed descriptions of the batchformula and methods used to prepare the encapsulated enteric coatedproanthocyanidin polymer composition bead formulation based on sugarspheres are provided below. Each hard-shell gelatin capsule contained250 mg proanthocyanidin polymer composition enteric coated beads.Capsules were packaged in HDPE bottles containing sixteen (16) 250 mgcaps each. The formulation for enteric coated proanthocyanidin polymercomposition beads contained 17.3% (w/w) of nonpareil seeds (sugarspheres 40/60 mesh, Paulaur, lot #60084060), 64.5% proanthocyanidinpolymer composition from C. lechleri, 1.5% hydroxypropylmethylcellulose(Methocel E5 Premium, Dow Chemical Co., lot #MM9410162E), 0.5% OpadryClear (Colorcon, lot #S83563), 14.5% “EUDRAGIT™ L 30D” (Rohm Tech., lot#1250514132), 1.45% triethyl citrate (Morflex, lot #N5×291), glycerylmonostearate (Imwitor-900, Rohm Tech, lot #502-229), and purified water(USP).

The layering coating solution containing the proanthocyanidin polymercomposition was prepared by adding hydroxypropylmethylcellulose and theproanthocyanidin polymer composition to purified water (USP) and mixinguntil dissolved. The nonpareil seeds were loaded into the product bowlof the fluid bed processor (Nior-Precision Coater). The polymer solutionwas then layered on the nonpareil seeds by spraying the solution ontothe fluidized nonpareil seeds at a target bed temperature of 30-35° C.Once the proanthocyanidin polymer layering had been completed, a sealcoat using Opadry Clear (preparing by mixing the Opadry Clear withPurified Water, USP) was applied with a target bed temperature of 30-35°C. After the seal coat was applied, the pellets were discharged andscreened through 1000μ and 425μ screens, and the layered spheres largerthan 425μ and smaller than 1000μ were charged back into the fluid bedprocessor. Meanwhile, the enteric coating solution was prepared bymixing triethyl citrate and glyceryl monostearate to water that had beenheated to 65° C. and then mixing this solution with the “EUDRAGIT™ L30D-55”. The resulting enteric coating solution was then sprayed ontothe layered spheres in the fluidized bed processor, at a bed temperatureof 30-35° C., until all the enteric coating solution was layered on thebeads. Based on the results of the HPLC assay indicating that theproanthocyanidin polymer composition waspresent at a concentration of52.9%, the enteric coated beads were hand filled into a Size #0 hardshell gelatin capsule to provide a 250 mg dosage and then packaged intoa suitable HDPE bottles with a heat induction lined cap.

TABLE 1 BATCH FORMULA Product: Proanthocyanidin Polymer Enteric CoatedBeads Batch Size: 578.0 gm Amount Used Raw Material Per Batch SugarNonpareil Spheres, NF (40/60) 100.0 gm Proanthocyanidin PolymerComposition 372.8 gm Hydroxypropylmethylcellulose E5, USP (K29/32) 8.7gm Opadry Clear (YS-1-19025A) 2.9 gm “EUDRAGITTM L 30D-55” (30% solids)279.4 gm Triethyl Citrate, NF 8.4 gm Glycerol Monostearate 1.4 gm Water,USP (Removed during processing) 1284.8 gm

1B. Encapsulated Enteric Coated Beads

Described below are the formula and methods used to prepare encapsulatedenteric coated bead formulations that do not contain nonpareil sugarspheres. One formulation contains 83.3% w/w proanthocyanidin polymercomposition, 0.5% w/w Opadry clear, 14.5% w/w “EUDRAGIT™ L 30D-55”, 1.9%w/w triethyl citrate and a 0.34% glyceryl monostearate.

The beads were first seal coated with a 5% solution of Opadry clear in a16 liter aeromatic MP-1 fluidized bed processor with a 50 mm Wurstercolumn. The coating parameters for the application of the seal coatingwere an inlet temperature of 50° C. to 60° C., an outlet temperature of25° C. to 40° C., an air volume of 30 to 40 CMH, a spray rate of 6 to 12grams per minute, and an air pressure of 2.5 Bar. After the seal coatwas applied, the beads were discharged and screened for beads largerthan 425μ and smaller than 1000μ. The beads of appropriate size werethen charged back into the fluid bed processes for enteric coating. Foreach 1000 grams of proanthocyanidin polymer composition beads, anenteric coating suspension was prepared from 811.97 grams “EUDRAGIT™ L30D-55”, 24.36 grams triethyl citrate, 4.36 grams glyceryl monostearateand 248.55 grams purified water. This suspension was prepared by gentlystirring the “EUDRAGIT™ L 30D-55” suspension continually and, in aseparate container, suspending and homogenizing the triethyl citrate andtalc in purified water. The triethyl citrate/talc mixture was then addedto the “EUDRAGIT™ L 30D-55” suspension, and the resulting coatingdispersion stirred during the spraying process to avoid settling. Thebeads were then coated in the fluidized bed processor under thefollowing parameters: The inlet temperature was 42° C. to 47° C.; theoutlet temperature was 28° C. to 34° C.; the air volume was 30-40 CMII;the spray rate was 6-12 grams/minute; and the air pressure was 2.5 Bars.The resulting enteric coated beads were then filled into a size #0 hardshell gelatin capsule.

1C. Enteric Coated Granules and Powder Particles

Described below is a method for formulating the proanthocyanidin polymercomposition as enteric coated granules or powder (microspheres with adiameter of 300-500μ) in either hard shell gelatin capsules or suspendedin an oral solution. The proanthocyanidin polymer composition powderparticles are prepared by high-shear powder mixing of theproanthocyanidin polymer composition and hydroxypropylmethylcellulose ina high speed mixer/granulator. The proanthocyanidin polymer compositiongranules are prepared by spraying polyvinylpyrrolidone on the powder inthe high speed mixer/granulator so that the powder particles agglomerateto form larger granules. Using fluidized bed equipment, the granules orpowder are then covered with a seal coat of Opadry Clear (mixed withwater) and then coated with the enteric coating “EUDRAGIT™ L 30D”applied as an aqueous dispersion containing 30% w/w dry methacrylatepolymer substance, which is supplied with 0.7% sodium lauryl sulfate NF(SLS) and 2.3% polysorbate 80 NF (Tween™ 20) as emulsifiers, to whichthe plasticizers, triethyl citrate and glyceryl monostearate, are addedto improve the elasticity of the coating. The final composition of theenteric coated powder is 81.8% w/w proanthocyanidin polymer composition,1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w“EUDRAGIT™ L 3013”, 1.45% w/w triethyl citrate, and 0.25% w/w glycerylmonostearate. The final composition of the enteric coated granules is81.8% w/w proanthocyanidin polymer composition, 10%polyvinylpyrrolidone, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/wOpadry clear, 14.5% w/w “EUDRAGIT™ L 30D”, 1.45% w/w triethyl citrate,and 0.25% w/w glyceryl monostearate.

The enteric coated proanthocyanidin polymer composition granules orparticles may be filled into a hard shell gelatin capsule in an amountwhich provides a suitable dosage.

The enteric coated proanthocyanidin polymer composition granules orpowder particles can also be suspended in a solution for oraladministration, particularly for pediatric administration. Thesuspension solution is prepared by wetting 2 gramshydroxypropylmethylcellulose in 97.8 ml distilled water and 0.2 gramsTween™ 80; mixing this preparation to homogeneity by sonicating, heatingthe solution to 40° C. and stirring for three hours; and then adding theenteric coated proanthocyanidin polymer composition powder particles orgranules to the homogeneous solution.

1D. Enteric Coated Compressed Tablets

A method for formulating the proanthocyanidin polymer composition with adiluent as enteric coated tablets is described below. For each 350 mgtablet, 250 mg proanthocyanidin polymer composition is granulated with 7mg crosslinked sodium carboxymethylcellulose (“AC-DI-SOL™”) and asufficient mass of microcrystalline cellulose (“AVICEL™ PH 200/300”) tobring the total mass to 350 mg. These ingredients are mixed for 20 to 30minutes in a V blender. After the 20 to 30 minutes of mixing, 1.75 mgmagnesium stearate is added and the mixture is blended for an additional4 to 5 minutes. The resulting granules are compressed on a rotary tabletpress using 5/16th inch standard concave punches. The tablets are coatedwith an enteric coating mixture prepared from 250 grams “EUDRAGIT™ L 30D-55”, 7.5 grams triethyl citrate, 37.5 grams talc and 205 grams water.The tablets are then placed in a perforated pan coater (e.g. the“ACCELA-COTA™” system) and rotated at 15 rpm at 40° C. The entericcoating formulation is sprayed using the following conditions: inlet airtemperature of 44° C.-48° C., exhaust air temperature of 29° C.-32° C.,product temperature of 26° C.-30° C., a 1 mm spray nozzle, a pan speedof 30 to 32 rpm, an airflow of 30-32 CFM, and a spray pressure of 20PSI. The tablets are finally cured for 30 minutes as the pan is rotatingat 15 rpm with an inlet air temperature of 60° C. and then, aftershutting off the heat, the tablets are rotated at 15 rpm until thetablets have cooled to room temperature.

1E. Enteric Coated Directly Compressed Tablets

A method for formulating the proanthocyanidin polymer compositionwithout a diluent as enteric coated tablets was carried out as describedbelow. Directly compressible proanthocyanidin polymer composition wasproduced according to the method described in Example 1F, infra. 125 mgtablets were prepared by blending 99.6% w/w directly compressibleproanthocyanidin polymer composition with 0.40% w/w magnesium stearatefor two minutes and then directly compressing the material into 125 mgtablets on a rotary press using ¼ inch diameter round standard concavepunches to a tablet hardness of 4-10 Kp.

The core tablets were tested and found to have an average hardness(n=10) of 4-10 Kp, friability (n=20) of less than 0.7%, an average tableweight (n=10) of 125 mg±7 mg, an average thickness (n=10) of 3.9 to 4.1mm, and a disintegration time (n=6) of not more than 20 minutes.

The coating dispersion was prepared by mixing, per 100 grams of tablets,22.22 grams of a 30% w/w “EUDRAGIT™ L 30D-55” suspension, kept gentlystirred with a mixture of 0.67 grams triethyl citrate, 1.67 grams talcand 20.44 grams purified water which had been mixed until homogeneous.The coating dispersion was continually stirred to avoid settling.

The tablets (in batches of 100,000) were coated with the coatingdispersion in a Compu-Lab 24 inch/30 L pan. The tablets were jogged inthe pan at a speed of 3-5 rpm and pre-warmed to a temperature of 35° C.to 40° C. The tablets were then coated with the enteric coatingdispersion to a 6% to 8% weight gain with the following parameters: aninlet temperature of 45° C. to 65° C.; an exhaust air temperature of 27°C. to 34° C.; a product temperature of 28° C. to 32° C.; a pan speed of8-14 rpm; an air flow of 180 to 240 CHM; an air spray pressure of 10-20psi (pounds per square inch); an initial spray rate of 3 to 4grams/min/kg; and a final spray rate of 4 to 8 grams/min/kg. The tabletswere then cured for 30 minutes in the pan with an inlet temperature of45° C. to 50° C. and a pan speed of 3 to 5 rpm. Finally, the tabletswere allowed to cool to room temperature in the pan at a pan speed of 3to 5 rpm. Four of the 125 mg tablets were then filled into a size zero,opaque Swedish orange-colored gelatin capsule.

The enteric coated proanthocyanidin polymer composition tablets weretested for content uniformity, drug release, microbiological tests andstability, and some analytical in process tests were also performed. Instability studies, the proanthocyanidin polymer composition remainedstable after six months of storage at room temperature as well as underaccelerated temperature and humidity conditions. Finally, the coretablets were tested and found to have an average hardness (n=10) of 4-10Kp, friability (n=20) of less than 0.7%, an average tablet weight (n=10)of 125 mg-±7 mg, an average thickness (n=10) of 3.9 to 4.1 mm, and adisintegration time (n=6) of not more than 20 minutes.

1F. Enteric Coated Directly Compressed Tablets

Formulation of the proanthocyanidin polymer composition, without adiluent, as enteric coated tablets was carried out as described below.The directly compressible proanthocyanidin polymer composition wasisolated as described in Example 2, infra. The core tablets wereprepared by milling 250 mg proanthocyanidin polymer composition pertablet (approximately 16 kg total) in a Quadro Comil with an 024R (30mesh) screen and then blending the milled composition in a PattersonKelley 2 cubic foot twin shell blender. 1 mg magnesium stearate(Spectrum Quality Products, Inc., New Brunswick, N.J.) per tablet wasthen added to the composition in the blender and blended for 2 minutes.The blend was then compressed into 251 mg tablets (containing 250 mgproanthocyanidin polymer composition) on a rotary tablet press to atablet hardness of 8-15 Kp and friability less than 0.5%.

The coating dispersion was prepared by first mixing in a first containerthe 25 g (7.5 g solids) “EUDRAGIT™ L 30 D-55” (Huls America, Inc.,Somerset, N.J.) (weight given per 115 grams coated tablets) dispersion.The pigment dispersion was prepared by adding sequentially with constantstirring in a second container 39.59 g purified water, 3.30 grams talc(Alphafil™ 500) (Whittaker, Clark & Daniels, Inc., South Plainfield,N.J.), 6.06 g (3.15 g solids) White Dispersion (pigment)(Wamer-Jenkinson, Inc., St. Louis, Mo.), and then 1.05 g triethylcitrate (Morflex, Inc., Greensboro, N.C.). The mixture was thenhomogenized for 15 minutes or until homogenous. While slowly stirring,the pigment dispersion was added to the “EUDRAGIT™ L 30 D-55” dispersionand then stirred for 30 minutes before spraying. Stirring was alsomaintained during the spraying process to avoid settling.

The tablets were coated in batches of 50,000 in a Compu-Lab 24 inch/30 Lpan with the following settings: 10-20 psi atomizing air pressure; 35°C.-60° C. pan inlet air temperature; 5 to 6 inches nozzle tip to tabletbed distance; and 4/2 baffles/nozzles. After adding the tablets to thepan, the pan was jogged at a speed of 3 to 5 rpm and heated to 40° C.The tablets were then sprayed to a weight gain of 11 to 13% with thefollowing parameters: 27°-33° C. target exhaust temperature (to beachieved within ten minutes of spraying); pan speed of 8 to 12 rpm;180-240 CFM air flow; and a spray rate of 2-5 g/min/kg. After achievingthe desired weight gain, the heat was shut off and the pan jogged at 3-5rpm until the tablets were cooled to below 30° C.

The tablets were encapsulated in size AA opaque Swedish orange coloredDB gelatin capsules (Capsugel, Greenwood, S.C.).

500 mg tablets were also produced as described above, except thatcoating was done on batches of 25,000 tablets to a weight gain of 8 to10%.

Example 2 Isolation of Directly Compressible Proanthocyanidin PolymerComposition

A directly compressible proanthocyanidin polymer composition (used toprepare the formulations in Examples 1E and 1F above) was isolated fromthe latex of the Croton lechleri plant as follows:

460 liters of Croton lechleri latex was mixed with 940 liters purifiedwater for ten minutes and then allowed to stand overnight (12 hours) at4° C. The red supernatant was pumped into a holding tank and the residuediscarded. The supernatant was then extracted with 200 liters n-butanolby mixing for ten minutes and then allowing the phases to separate. Then-butanol phase was discarded, and the aqueous phase was extracted twomore times with 200 liters n-butanol each time. After extraction, theaqueous phase was concentrated by ultrafiltration using a 1 kD cut-offmembrane (a low protein binding cellulose membrane), and then theretentate was dried in a tray dryer at approximately 37° C. (±2° C.).

For purification by column chromatography, 6 kg of the dried extract wasdissolved in 75 liters of purified water and stirred for 90 minutes. Thedissolved material was chromatographed on a two column chromatographysystem consisting of a 35 liter CM-Sepharose column (a weak cationexchange resin) and a 70 liter LH-20 column (a size-exclusion resin)connected in series. The material was loaded onto the CM-Sepharosecolumn, washed with 140 liters purified water, and then eluted onto theLH-20 column with 375 liters of 30% acetone. At this point, the twocolumns were disconnected, and the proanthocyanidin polymer compositionwas eluted from the LH-20 column with 250 liters of 45% liters acetone.Fractions were collected into 10 liter bottles and monitored with a UVdetector at 460 nm. Fractions containing material having detectableabsorbance at 460 nm were pooled and concentrated by ultrafiltrationusing a 1 kD cut-off membrane (a low protein binding cellulosemembrane). The retentate was dried using a rotary evaporator in awaterbath at approximately 37° C. (±2° C.).

The proanthocyanidin polymer composition was tested for directcompressibility. 250 mg portions of the proanthocyanidin polymercomposition, in the absence of any binders or excipients, was placedinto a tableting machine and then pressed into tablets of varyingthicknesses (i.e., the greater the pressure on the composition to formit into a tablet, the thinner the resulting tablet). The hardness of thetablets was then determined in a conventional hardness tester. Thefriability of tablets having a hardness of 8-15 kp was determined asdescribed in USP 23<1216>. The friability was less than 0.5% loss inweight.

Example 3 Components, Composition, and Manufacturing of a Drug Product

3A. Drug Product

The drug product, crofelemer, consists of an enteric-coated 125 mgtablet(s) over-encapsulated in a Size 00, opaque Swedish orange gelatincapsule and backfilled with microcrystalline cellulose. Each capsulecontains 1, 2, or 4 enteric-coated tablets. The tablet core consists of99.93% crofelemer and 0.07% magnesium stearate and is coated with amethacrylic acid copolymer.

3B. Components of Drug Product

Crofelemer is supplied by Napo Pharmaceuticals, Inc., and ismanufactured under current Good Manufacturing Practices (cGMP).Magnesium stearate is manufactured by Mallinckrodt (or equivalent) andmeets the specifications for magnesium stearate as described in 27USP/NF. The magnesium stearate is certified by the manufacturer to bederived from vegetable sources. Microcrystalline cellulose ismanufactured by FMC (or equivalent) and meets the specifications formicrocrystalline cellulose as described in 27 USP/NF. Both high-densityand low-density microcrystalline cellulose are employed. Methacrylicacid copolymer is manufactured by DeGussa under the trade name Eudragit®(L30-D55) and meets the specifications for methacrylic acid copolymer,Type C, as described in 27 USP/NF. Triethyl citrate is manufactured byMorflex (or equivalent) and meets the specifications for triethylcitrate as described in 27 USP/NF. Talc is manufactured by Whittaker,Clark and Daniels (or equivalent) and meets the specifications for talcas described in 27 USP/NF. Purified water is supplied by the drugproduct manufacturer and meets the specifications for purified water asdescribed in 27 USP/NF. Swedish orange opaque, Size 00, hard gelatincapsule bodies and caps are supplied by Capsugel, Inc. (Greenwood,S.C.). The manufacturer certifies that the capsules are made fromgelatins that meet the current National Formulary (NF) requirements forgelatin under cGMP.

3C. Composition of Drug Product

The composition of the tablet cores and the enteric-coated tablets isdescribed in Table 2 and Table 3, respectively. The amount of crofelemerand magnesium stearate is adjusted based on the anhydrous potency of thedrug substance, which corrects for the moisture content of thecrofelemer. The amount of weight gain after coating is approximately10%. The clinical batch size ranges from 100,000 to 150,000enteric-coated tablets. Subsequently, 1, 2, or 4 enteric-coated tabletsare placed in a Size 00 capsule and backfilled with microcrystallinecellulose to match weights of each capsule strength and placebo.

TABLE 2 Composition of Drug Product 125 mg Tablet Cores QuantitativeTheoretical Composition mg/unit Ingredient Grade Purpose (w/w %) doseCrofelemer cGMP Active 99.93% 125 mg Magnesium 27 USP/NF Lubricant 0.07% 0.13 mg stearate Total  100% 125.13 mg

TABLE 3 Composition of Drug Product Enteric-Coated 125 mg TabletsQuantitative Theoretical Composition mg/unit Ingredient Grade Purpose(w/w %) dose Crofelemer tablet cGMP Active 90.0% 125.13 mg Eudragit L-30D55 27 USP/NF Coating  7.4% 34.5 mg (10.4 mg solids) Triethyl citrate 27USP/NF Plasticizer  0.8% 1.05 mg Talc 27 USP/NF Dispersing  1.8% 2.6 mgPurified water* 27 USP/NF Solvent N/A* 21.9 mg Total  100% 136.4 mg*Purified water is removed during process.

3D. Method of Manufacturing the Drug Product

I. Manufacture of Tablet Core

A sufficient amount of crofelemer and magnesium stearate, based on thepotency of crofelemer, on an anhydrous basis that adjusts for the amountof moisture, is staged prior to manufacture. Crofelemer is added to theblender and magnesium stearate is screened and added to the crofelemer.The crofelemer and magnesium stearate are blended, a representativeblend sample is taken, and the yield is determined. Yield must bebetween 100±3%. Blend uniformity is not determined, except as needed,because the blend is 99.9% active. The blend is directly compressed on arotary tablet press, using '/4 inch round concave punches. Finishedtablet cores are de-dusted and placed in a container to await coating.Prior to the start of the manufacturing run, pre-production runs areperformed to adjust the speed and compression of the press in order tomeet the targeted tablet-core weight, thickness, and hardness. Inaddition, friability and disintegration are measured. During themanufacturing run, tablet-core samples are taken at periodic intervalsto ensure that the tablets continue to meet the targeted tablet weight,thickness, and hardness. Representative tablet cores are taken duringthe beginning, middle, and end of the production run for additionaltesting for hardness, thickness, weight, friability, and disintegration.The average tablet-core weight must be within ±5% of the targetedtablet-core weight. The number of tablet cores, sample tablet cores,tablet-core waste, and blend waste are reconciled and the percentaccountability calculated. The percent accountability must not be lessthan 95% and not more than 103%. A schematic of the tablet coremanufacturing process is presented below.

Schematic of Tablet Core Manufacturing Process Weigh crofelemer ↓ Weighmagnesium stearate ↓ Blend ↓ Compress tablets ↓ De-dust ↓ Collect tabletcores for coating

II. Coating of Tablet Core

The amount of Eudragit®, triethyl citrate, talc, and purified water iscalculated and staged based on a nominal tablet-core weight gain of 10%.Purified water is charged into a suitable container equipped with ahigh-shear mixer. The triethyl citrate and talc are added to thecontainer and mixed until homogenized. In a separate container equippedwith a propeller mixer, the Eudragit® dispersion is charged and mixed.The triethyl citrate and talc dispersion is added to the Eudragit®dispersion and is continuously stirred throughout the spraying process.The pan-coater machine parameters are adjusted as appropriate and thelines are charged with the coating dispersion. The tablet cores arecharged in a coating pan and warmed to 35 to 40° C. while jogging thecoating pan. Once at temperature, the average weight of the tablet coresis recorded and the targeted coated tablet weight is calculated.Subsequently, the tablet cores are sprayed and periodicallyweight-checked until the targeted weight gain is met. The targeted sprayrate (4 to 8 g/min/kg of tablet cores), the targeted exhaust temperature(35 to 40° C.), and inlet temperature are monitored at frequentintervals. The tablets are cured for 30 minutes at 45 to 50° C., andthen cooled. Representative enteric-coated samples are taken fortesting. The average enteric-coated tablet weight must be within ±5% ofthe targeted enteric-coated tablet weight.

The weight of enteric-coated tablets, enteric-coated tablet samples, andtheoretical quantity of enteric-coated tablets are determined and thepercent accountable yield calculated. The percent accountable yield mustnot be less than 95% and not more than 103%. The tablet-corespray-coating process is illustrated below.

Schematic of Tablet-Core Spray-Coating Process Add triethyl citrate andtalc to purified water ↓ Homogenize ↓ Mix Eudragit ® dispersion ↓ Addtriethyl citrate and talc dispersion to Eudragit ® dispersion ↓ Mix ↓Add tablet cores to pan coater ↓ Heat to 35-40° C. (exhaust temp) ↓Spray tablet cores (4-8 g/min/kg of tablet cores) until targeted weightgain is reached ↓ Cure at 45-50° C. ↓ Cool ↓ Collect enteric-coatedtablets for over-encapsulation I

III. Manufacture of Over-Encapsulated Enteric-Coated Tablets

The enteric-coated tablets and microcrystalline cellulose are stagedseparately for the over-encapsulation of each capsule strength. Theamount of microcrystalline cellulose to be encapsulated is calculated inorder to achieve a nominal 600 to 800 mg capsule weight, dependent uponthe exact dosage form (125 mg, 250 mg, or 500 mg). The average weight ofthe capsules is calculated based on an average of 100 capsules. Thecapsules are filled using a semi-automatic over-encapsulation machinefitted with Size 00 change parts and adjusted to deliver the properamount of tablets and microcrystalline cellulose. Pre-production runsare performed in order to adjust the tray fill weight, the Add tabletcores to pan coater number of turns on the tamper, and the number oftimes tamped in order to meet the targeted gross capsule weight (capsuleshell plus tablets and microcrystalline cellulose). Each tray isprepared by placing the capsule bodies in the capsule tray. Each capsulebody is filled with the targeted amount of tablets and microcrystallinecellulose to achieve the targeted fill. The caps are placed on thebodies and closed. The capsules are removed from the tray and de-dusted.A composite of capsules from each tray is collected and weighed forin-process and release testing. The average capsule weight is within ±5%of the targeted capsule weight. The process is then repeated until thedesired number of capsules is filled. Damaged capsules, enteric-coatedtablet waste, and microcrystalline cellulose waste are collected forfinal product reconciliation. A composite of the finished capsules issent for release testing. The number of finished capsules, samplecapsules, damaged capsules, and drug substance waste are reconciled andthe percent accountability is calculated. The percent accountabilitymust not be less than 95% and not more than 103%. A schematic of theover-encapsulation of the enteric-coated tablets is presented below.

Schematic of Over-Encapsulation of Enteric-Coated Tablets Weighmicrocrystalline cellulose and enteric-coated tablets Place capsulebodies in over-encapsulation machine Fill capsule bodies with tablet(s)and microcrystalline cellulose Cap capsule bodies and remove capsulesDe-dust

Example 4 Effect of Enterically Coated Proanthocyanidin PolymerComposition on Patients Suffering from Diarrhea Predominant IrritableBowel Syndrome

4A. Treatment

The study was a 16-week, multi-center, Phase 2, randomized,double-blind, placebo controlled study in subjects with diarrheapredominant irritable bowel syndrome (d-IBS). Two-hundred and forty-six(246) subjects, meeting the definition of d-IBS as supported by the Rome11 Criteria for the Diagnosis of IBS were randomized into four groups:placebo, 125 mg bid, 250 mg bid, and 500 mg bid. The study consisted ofa 2-week Determine average weight of capsules treatment-free screeningperiod, a 12-week blinded treatment period, followed by a 2-weektreatment-free follow-up period.

During the 2-week screening period, subjects self-reported dailyinformation about the status of their d-IBS symptoms via a touch-tonetelephone diary. This included information about abdominal pain anddiscomfort, stool frequency, consistency and urgency. If the subjectcontinued to meet the inclusion criteria at the end of the screeningperiod, and the information captured in the diary during the screeningperiod indicated they had active d-IBS [mean daily stool frequency≧2;pain score≧1; stool consistency≧3 (5-point Lickert scale for pain andconsistency)], subjects were randomized into one of four groupsaccording to a computer-generated central randomization schema.

During the 12-week double-blind treatment period, subjects continued torecord daily and weekly assessments via a touch-tone telephone diarysystem as instructed. Subjects were seen every 4 weeks during thetreatment period for study assessment visits at which time they receivedadditional study medication.

During the 2-week treatment-free follow-up period, subjects continued torecord daily and weekly assessments.

Results:

There were no drug-related serious adverse events. Adverse event rateswere similar across all dose groups as shown in Table 4. There were nodrug- or dose-related differences in constipation.

TABLE 4 Summary of Adverse Events¹ 125 mg 250 mg 500 mg Placebo bid bidbid No. Subjects/Group 61 62 59 62 No. Subjects with ≧ 1 AE 14 12 15 15No of GI AEs 7 10 10 10 GI Event Abdominal distention 0 1 0 1 Abdominalpain 1 2 1 2 Abdominal tenderness 0 0 1 0 Constipation 1 3 2 1 Diarrhea1 2 1 0 Dry mouth 0 0 0 2 Eructation 0 0 0 1 Flatulence 1 1 3 1Haematochezia 0 0 1 0 Hemorrhoids 0 1 0 0 Nausea 3 0 1 1 Urgency 0 0 0 1No. of Other AEs 11 12 15 14 Other Events (>1 AE/group) Dizziness 1 0 30 Headache 2 2 1 3 Anxiety 2 0 0 1 Insomnia 0 0 2 1 Rash 1 2 0 0¹possible, probably, or likely related.

In general, crofelemer 125 mg bid, as shown in Table 5 exhibited aconsistent response among most efficacy endpoints in females. Thereappeared to be little efficacy in males; however, group size was toosmall (13-16/group) to analyze separately. Since crofelemer produced noconstipation, stool consistency scores approached normal and were notdifferent from placebo. In all other endpoints (frequency, urgency,adequate relief, and pain), there was an improvement in activity witheach successive month and during the 2 week treatment-free follow-upperiod symptoms began approaching baseline levels as expected.Crofelemer 500 mg bid also had a statistically significant effect onpain (0.48 decrease in pain score; 22.44% increase in pain free days).There were 5 female disease outliers (they had >9 stools/day at baselineand were >3 standard deviations from the mean stool frequency of allrandomized female subjects) that were not representative of the d-IBSpopulation used in this study and were removed from all analysespresented in this summary.

As shown in Table 6, crofelemer at 250 mg and 500 mg bid had less of aneffect on frequency and consistency than the placebo group. Subjectstreated with crofelemer 500 mg bid had a greater than half of stool perday mean increase in stool frequency as compared to placebo; there meanstool consistency was 0.22 points higher (closer to loose consistency)as compared to placebo.

TABLE 5 Efficacy of Crofelemer 125 mg bid in Females Endpoint (A fromPlacebo) Results¹ Consistency Score −0.03 Frequency (stools/day) −0.7Urgency Free Days 11.2% Pain score −0.42* Pain Free Days 12.76%*Adequate Relief 16% ¹Month 3 results; observed case analysis withdisease outliers (mean baseline frequency >9 stools/day) removed fromall groups, ‘statistically significant at p < 0.05

TABLE 6 The Effects of Crofelemer.250 and 500 mg bid on stoolconsistency and frequency Endpoint (A from Placebo) 250 mg bid¹ 500 mgbid¹ Consistency Score 0.19 0.22 Frequency (stools/day) 0.24 0.60 ¹Month3 results: observed case analysis with disease outliers (mean 1 >9stools/day) removed from all groups.

As seen in FIG. 4, female subjects treated with crofelemer 125 mg bidhad a clinically significant decrease in stool frequency. Use ofcrofelemer led to a decrease of greater than one bowel movement per day.The month-to-month improvement in stool frequency was contrasted by theplacebo effect diminishing at month two as values began to approachbaseline. When the treatment-free period began at the end of monththree, the subjects stopped taking crofelemer and the effect began to goaway as expected.

As seen in FIG. 5, crofelemer produced an increase in urgency free days.At Month 3, there was a 35.1% increase in urgency free days in thecrofelemer group versus a 23.9% increase in urgency free days observedin the placebo group. There was a month-to-month improvement, ascommonly seen. The placebo effect peaked at month two and abruptlydecreased as values begin to approach baseline. When the treatment-freeperiod began at the end of month three, the subjects stopped takingcrofelemer and the effect began to go away as expected.

As seen in FIG. 6, administration of crofelemer produced an increase inthe percentage of subjects reporting adequate relief of d-IBS symptoms.Since d-IBS is comprised of a group of symptoms, the adequate reliefendpoint is an overall assessment by the patient as to how well thetreatment is addressing their d-IBS symptoms. Crofelemer at 125 mg bidcaused a 16% increase in the adequate relief of d-IBS symptoms ascompared to the placebo group. With the observed analysis, there was amonth-to-month improvement in activity of crofelemer, i.e., the longerthe patient took crofelemer, the greater relief of symptoms. Aspreviously seen with other endpoints, the placebo effect peaked at monthtwo and decreased thereafter.

Diarrhea-predominant IBS is differentiated from many functional boweldiseases by the close association of pain with changes in bowel habits.As defined by the Rome TI Criteria for the Diagnosis of IBS, pain mustbe associated with abnormal bowel habits and the improvement of thebowel habits should be associated with the improvement of pain. In thisstudy we have measured pain score with a 5-point scale, where 0 is noneand 5 is severe; and the presence of pain free days. As shown in FIG. 4and FIG. 5, female subjects treated with crofelemer (125 and 500 mg bid)had clinically and statistically significant (p<0.05) decreases in bothpain score and pain free days. The effect is quite consistent asobserved in the weekly pain score results shown in FIG. 6. This effecton pain relief was unexpected and surprising.

In conclusion, crofelemer at 125 mg bid, was safe and its administrationresulted in improvement in the efficacy endpoints of pain, adequaterelief, frequency, and urgency in female subjects. Crofelemer at 250 and500 mg bid was safe and, compared to placebo, appeared to worsen thediarrheal symptoms of consistency and frequency. Crofelemer at 125 and500 mg bid produced a statistically significant decrease in both painscore and pain free days in female subjects with d-IBS.

Example 5 Investigation of Crofelemer Mechanism of Action

To further investigate the mechanism of action of crofelemer, crofelemerat 10 and 100 μg/mL was evaluated in a selected panel of cellularcytokine release assays (IL-1α; TNF-α-induced and IL-1-induced PGE2release; IFN-γ; IL-2, IL-4; IL-5; IL-6; IL-8; IL-10 and TNF-α) andmolecular assays (COX-1 and COX-2 enzyme assays, and glucocorticoid,serotonin 5HT3, δ-opiod, κ-opiond, μ-opiod, and non-selective opiodreceptor binding assays. Crofelemer was also tested in cytotoxicityassays corresponding to the ConA- and LPS-induced cytokine releaseassays as well as those corresponding to the TNF-a- and IL-1-induced PGErelease assays.

At both 10 and 100 μg/mL, crofelemer caused a 73% and 100% inhibition inthe COXI and COX2 enzyme assays, respectively.

Further, crofelemer exhibited significant (>50%) inhibition in theIFN-γ; IL-2, IL-4; IL-6; IL-8; IL-10 and TNF-a cytokine release assaysas well as in the TNF-α-induced and IL-1-induced PGE2 release assays at10 and 100 μg/mL (i.e., all cytokine release assays with the exceptionof IL-5). Crofelemer also displayed significant cytotoxic activity inthe ConA and IL-1-induced PGE2 cytotoxicity assays suggesting that inthe ConA-mediated cytokine release assays (IFN-γ, IL-2, IL-4 and IL-10)and the IL-I-induced PGE2 release assay may be due to generalcytotoxicity.

Example 6 Animal Toxicity Study

In an animal toxicity study, crofelemer was demonstrated to producediarrhea in dogs at dosages of 175 mg/kg/day and 600 mg/kg/day.

In a separate example, diarrhea was induced in healthy human volunteersin a phase I study at a dosage of 2000 mg/day.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled. Such modifications areintended to fall within the scope of the appended claims.

All references, patent and non-patent, cited herein are incorporatedherein by reference in their entireties and for all purposes to the sameextent as if each individual publication or patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety for all purposes.

1. A method of treating constipation-predominant irritable bowelsyndrome (c-IBS), comprising administering to a patient in need of suchtreatment, an amount of an extract or latex isolated from Croton spp. orCalophyllum spp. effective to treat c-IBS.
 2. A method of treatingconstipation-predominant irritable bowel syndrome (c-IBS), comprisingadministering to a patient in need of such treatment, an amount of apolymeric proanthocyanidin composition isolated from Croton spp. orCalophyllum spp. effective to treat c-IBS.
 3. The method of claim 1 or2, in which said amount is bioequivalent to an orally administered doseof about 250 mg per day to about 4000 mg per day of crofelemer.
 4. Themethod of claim 2, in which the polymer composition comprises at leastone monomer selected from the group consisting of catechin, epicatechin,gallocatechin and epigallocatechin.
 5. The method of claim 2 wherein thepolymeric proanthocyanidin composition is crofelemer.
 6. The method ofclaim 5, in which crofelemer is orally administered in an amount ofbetween about 250 mg per day and about 4000 mg per day.
 7. The method ofclaim 1 or 2, in which the treatment comprises reducing one or moresymptoms of c-IBS.
 8. The method of claim 7, in which the symptom ispain or abdominal discomfort.
 9. The method of claim 8, in which pain ordiscomfort is reduced by at least 10%.
 10. The method of claim 7, inwhich the symptom is constipation or less than normal stool frequency.11. The method of claim 10, in which stool frequency is increased by atleast one additional bowel movement per week.
 12. A method of treatingalternating constipation-predominant/diarrhea-predominant irritablebowel syndrome (a-IBS), comprising administering to a patient in need ofsuch treatment, an amount of an extract or latex isolated from Crotonspp. or Calophyllum spp. effective to treat a-IBS.
 13. A method oftreating alternating constipation-predominant/diarrhea-predominantirritable bowel syndrome (a-IBS), comprising administering to a patientin need of such treatment, an amount of a polymeric proanthocyanidincomposition isolated from Croton spp. or Calophyllum spp. effective totreat a-IBS.
 14. The method of claim 12 or 13, in which said amount isbioequivalent to an orally administered dose of about 50 mg per day toabout 4000 mg per day of crofelemer.
 15. The method of claim 14, inwhich said amount bioequivalent to an orally administered dose of about500 mg per day to about 1500 mg per day of crofelemer.
 16. The method ofclaim 13, in which the polymeric proanthocyanidin composition iscrofelemer.